Project 4, Abstract Like all bacteria, Mycobacterium tuberculosis (Mtb) must coordinate major cellular processes, such as DNA replication, with the synthesis and segregation of structural components in order to grow and divide. While the processes governing cell division are reasonably well-described in model organisms, the mechanisms used by mycobacteria are clearly different and less well understood. Defining the mechanisms governing the essential process of cell division in Mtb will reveal new antimicrobial targets and provide the basis to understand how this process is regulated during the slow-growing states that contribute to bacterial persistence during infection. This project supports the program?s overall goal to understand pathways important to Mtb?s adaptation to disease-relevant stress. It will synergize with other projects and leverage each of the cores. Coordinating the cytokinesis with the cell cycle and the distribution of cellular material must be both temporally and spatially regulated. Work in the project labs has shown that this coordination requires at least three distinct regulatory paradigms. (1) The ordered assembly of the cell division complex, or ?divisome? requires spatial and temporal cues that may be provided by Ser/Thr protein kinases (STPK). (2) Extracytoplasmic enzymes responsible for cell wall metabolism represent a distinct regulatory challenge, and are often controlled by protein- protein interactions in the periplasmic space. (3) The coordination of processes necessary for cell division are not restricted to the septum; fundamental metabolic changes are also likely necessary to provide the precursors required for this major cellular event. The goal of this project is to understand the regulatory paradigms that control mycobacterial cell division. Specifically, the project will: Aim 1. Characterize the role of protein phosphorylation in divisome dynamics. Synchronized Mtb cultures, quantitative imaging, and biochemical approaches will be used to mechanistically characterize the role of phosphorylation in the temporal and spatial regulation of divisome function. Aim 2. Define and characterize extracytoplasmic complexes necessary for cell division. A combination of genetic and physical approaches will be used to find interactions that are important for regulating enzymatic activity and localization during cell division. Aim 3. Characterize the links between cell division and metabolism. A combination of metabolomics and genetics will be used to investigate the primary metabolic pathways necessary for cell division.